Nitric oxide is required for, and promotes auxin-mediatedactivation of, cell division and embryogenic cell formation butdoes not influence cell cycle progression in alfalfa cell cultures

Krisztina Otvos1, Taras P. Pasternak2,†, Pal Miskolczi2, Monika Domoki1, Dulguun Dorjgotov1, Attila Sz}ucs1, Sandor Bottka2,

Denes Dudits2 and Attila Feher1,*

1Laboratory of Functional Cell Biology, and2Laboratory of Cell Division and Differentiation, Institute of Plant Biology, Biological Research Centre, Hungarian Academy of

Sciences, Temesvari krt. 62, H-6726, Szeged, Hungary

Received 3 May 2005; revised 15 June 2005; accepted 17 June 2005.*For correspondence (fax 36 62 433 434; e-mail fehera@brc.hu).†Present address: Institute of Biologie II, Ludwig-Albert University, Sonnerster 5, 79104 Freiburg, Germany.

Summary

It is now well established that nitric oxide (NO) serves as a signaling molecule in plant cells. In this paper

experimental data are presented which indicate that NO can stimulate the activation of cell division and

embryogenic cell formation in leaf protoplast-derived cells of alfalfa in the presence of auxin. It was found that

various NO-releasing compounds promoted auxin-dependent division (as shown by incorporation of

bromodeoxyuridine) of leaf protoplast-derived alfalfa cells. In contrast, application of NO scavenger or NO

synthesis inhibitor inhibited the same process. Both the promotion and the inhibition of cell cycle activation

correlated with the amount and activity of the cognate alfalfa p34cdc2 protein Medsa;CDKA;1,2. The effect of

L-NG-monomethyl-L-arginine (L-NMMA) was transient, and protoplast-derived cells spending more than

3 days in culture become insensitive to the inhibitor as far as cell cycle progression was concerned. L-NMMA

had no effect on the cell cycle parameters of cycling suspension-cultured cells, but had a moderate transient

inhibitory effect on cells re-entering the cell cycle following phosphate starvation. Cycling cultured cells,

however, could respond to NO, as indicated by the sodium nitroprusside (SNP)- and 2-phenyl-4,4,5,5-

tetramethylimidazoline-1-oxyl-3-oxide (PTIO)-dependent accumulation of the ferritin protein. Based on these

observations, it is hypothesized that L-NMMA-sensitive generation of NO is involved in the activation, but not

the progression of the plant cell division cycle. In addition, SNP promoted and L-NMMAdelayed the exogenous

auxin [2,4-dichlorophenoxyacetic acid (2,4-D)] concentration-dependent formation of embryogenic cell

clusters expressing the MsSERK1 gene; this further supports a link between auxin- and NO-dependent

signaling pathways in plant cells.

Keywords: nitrogen monoxide, bromodeoxyuridine (BrdU) incorporation, Medicago sativa, protoplast,

somatic embryogenesis, 2,4-dichlorophenoxyacetic acid.

Introduction

Nitric oxide (NO) is a small, water- and lipid-soluble free

radical known to act as a biological messenger in animals.

NO ultimately exerts its biological effects by reacting,

either directly or through other reactive nitrogen interme-

diates, with a variety of targets such as heme groups, Fe–S

or Zn–S clusters, sulfhydryl groups or various other

chemical substrates (Cooper, 1999; Wink and Mitchell,

1998). That is why both metal- and thiol-containing

proteins are target sites for NO, and as such, are very

important cellular vehicles for the orchestration of the

biological functions of NO. Among these cellular target

proteins are cellular messengers, ion channels, enzymes,

receptors and transcription factors (Bogdan, 2001). The

diversity of potential targets is reflected by the large

number of different cellular systems that use NO as a

signal mediator. In mammals, NO was shown to be

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The Plant Journal (2005) 43, 849–860 doi: 10.1111/j.1365-313X.2005.02494.x

involved in signal transduction pathways controlling,

among others, responses to infection, apoptosis, cell pro-

liferation, differentiation and fertilization (Kuo et al., 2000;

Peunova et al., 1996; Schmidt and Walter, 1994).

In mammals, formation of NO mainly relies on the activity

of the enzyme nitric oxide synthase (NOS; EC 1.14.13.39).

NOS enzymes catalyze the formation of NO from L-arginine,

which undergoes oxidation to citrulline. In human cells,

three NOS isoforms have been identified with altered tissue

specificity and Ca2þ dependence (Wang and Marsden, 1995).

Numerous experiments have demonstrated the generation

and presence of NO within plant cells and tissues (Beligni

and Lamattina, 2001; Durner and Klessig, 1999; Lamattina

et al., 2003). Although the Arabidopsis genome does not

contain genes coding for the plant homologs of the mam-

malian NOS enzymes, a plant protein with significant

biochemical similarity to animal NOS has recently been

identified by reverse genetics (Guo et al., 2003). Involvement

of this enzyme in hormonal signaling has also been dem-

onstrated (Guo et al., 2003).

Although NOS-like enzymes have only recently been

discovered in plants, considerable amounts of experimental

data had already been accumulated on the role of NO as a

signaling molecule in plant defense mechanisms (Delle-

donne et al., 1998; Durner and Klessig, 1999) as well as in

abiotic stress responses (Gould et al., 2003; Mackerness

et al., 2001; Mata and Lamattina, 2001). In addition, several

observations indicate that NO is also involved in the

regulation of plant growth and development under normal

growth conditions. It was shown, for example, that NO

affects germination (Beligni and Lamattina, 2000; Leshem

and Pinchasov, 2000; Seregelyes et al., 2003), leaf expansion

(Zhang et al., 2003), senescence (Leshem and Haramaty,

1996), programmed cell death (Clarke et al., 2000; Durzan

and Pedroso, 2002; de Pinto et al., 2002) and organogenesis

(Correa-Aragunde et al., 2004; Pagnussat et al., 2002). Con-

cerning its possible methods of action, NO was demonstra-

ted to modulate signal transduction pathways in plant cells

by affecting the level of secondary messenger molecules

such as cyclic guanosine monophosphate (cGMP), cyclic

ADP-ribose (cADPR) or Ca2þ (Correa-Aragunde et al., 2004;

Durner and Klessig, 1999; Durner et al., 1998; Pagnussat

et al., 2003, 2004) and activating kinase cascades (Pagnussat

et al., 2004).

The interaction of NO-mediated signaling with the regu-

lation of cell division and differentiation in mammalian cells

is well established (Peunova et al., 1996). Nitric oxide has

been shown to interact with cell cycle regulation at various

levels, resulting in blocked cell division (Guo et al., 1998;

Ishida et al., 1997; Pervin et al., 2001; Takagi et al., 1994). In

plant cells, however, there are hardly any data on either the

direct or indirect involvement of NO in signal transduction

events related to the cell cycle or cell division (Pagnussat

et al., 2004).

Here, experiments are reported which indicate that NO

may influence entry into the cell cycle but not cell cycle

progression in cultured plant cells. In addition, the involve-

ment of NO in the formation of embryogenic cells from leaf

protoplasts is also demonstrated.

Results

Frequency of incorporation of bromodeoxyuridine (BrdU)

into cells derived from leaf protoplast is affected by NO

To investigate the possible effects of NO on the division of

protoplast-derived cells, the frequency of incorporation of

bromodeoxyuridine (BrdU) into nuclear DNA was deter-

mined in cultures grown in the presence of NO-releasing

compounds [(�)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-

hexeneamide (NOR-3), 3-morpholinosydnonimine (SIN-1),

N-acetyl-3-(nitrosothio)-D-valine (SNAP), sodium nitroprus-

side (SNP), 4-phenyl-3-furoxancarbonitrile (furoxan)], the

NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-

oxyl-3-oxide (PTIO) and the NOS inhibitor L-NG-mono-

methyl-L-arginine (L-NMMA). The same concentrations were

used as accepted for the treatment of animal cells. In

Figure 1 it is demonstrated that while the NO-donor com-

pounds increased the number of cells incorporating BrdU

into their nuclei during a 24-h period, both the NOS inhibitor

and the NO scavenger inhibited this process.

It can also be seen that the effect of the NO-releasing

compounds was concentration dependent: 10 lM concen-

trations promoted but 100 lM concentrations inhibited the

increase in S-phase frequency. NOR-3 at a concentration of

100 lM and furoxan at 10 and 100 lM were even deleterious

to the cells (data not shown).

Figure 1. Effect of NO donors (NOR-3, SIN-1, SNAP, SNP), scavenger (PTIO)

and inhibitor (L-NMMA) on relative frequency of incorporation of BrdU into

the nuclei of leaf protoplast-derived alfalfa cells. The cells were grown in the

presence of the indicated concentrations of 2,4-D and drug from the

beginning of culture. BrdU was applied to protoplast-derived cells for 24 h

at the time of entering into the S-phase (checked by flow cytometry). Relative

values in comparison with cells grown in the presence of 1 lM 2,4-D (100%)

are shown as the average of three repeats with standard deviation. As a

positive control we used 10 lM 2,4-D in the medium.

850 Krisztina Otvos et al.

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 43, 849–860

Attempts have also been made to determine cellular

NO levels in control and treated cells using a micro-

scopic approach based on the NO-sensitive fluorescent dye

4,5-diaminofluorescein diacetate (DAF-2DA) (Kojima et al.,

1998), allowing the detection of NO levels in individual

cells. DAF2-DA is a cell-permeable molecule that does not

fluoresce until it reacts with NO. Although we could

observe a DAF-2DA-dependent fluorescent signal in a

fraction of protoplast-derived cells, its NO-insensitive ana-

log, 4-aminofluorescein diacetate, exhibited very bright

fluorescence in all cells and this finding brought into

question the specificity of DAF-2DA (Beligni et al., 2002)

(see Figure S1).

The NO-donor sodium nitroprusside (SNP) promotes auxin

concentration-dependent division and embryogenic cell

formation in alfalfa leaf protoplast cultures

As shown in Figure 1, the effect of NO donors on the S-phase

frequency of protoplast-derived cells is very similar to the

effect of increased 2,4-dichlorophenoxyacetic acid (2,4-D)

concentration. In order to gain further insight into the effect

of NO donors and 2,4-D on leaf protoplast-derived cells,

various cellular parameters have been determined on cells

grown in the absence and presence of 10 lM SNP at various

2,4-D concentrations (0, 0.22 or 1 lM). As a control we used

‘inactive’ SNP (iSNP), pre-exposed to direct light for 1 day in

order to ensure complete decomposition preceding its

application to the protoplasts, at the same concentration.

This concentration of iSNP had no significant effect on the

development of protoplast-derived cells in comparison with

untreated cultures (data not shown).

In the absence of exogenous auxin, 10 lM SNP did not

influence cell division, as shown in Figure 2. However, in the

presence of either 0.22 or 1 lM 2,4-D, 10 lM SNP increased

the frequency of viable and dividing cells (Figure 2a,c).

Consistent with this observation, the amount and activity of

Medsa;CDKA;1,2 kinase was elevated in protoplast-derived

cells that were grown in the presence of 0.22 or 1 lM 2,4-D

and 10 lM SNP in comparison with untreated controls (2,4-D

only) (Figure 2b).

Cell morphology was also altered by this concentration of

active SNP. In the presence of 1 lM 2,4-D, for example, cell

elongation was considerably reduced (Figure 2d) and spher-

ical cells with dense cytoplasm were developed from these

protoplasts (Figure 2e). Previous studies showed that alfalfa

(a) (b)

(c) (d)

(e) (g)

(f)

Figure 2. The effect of the NO donor SNP on cell

division and embryogenic cell formation in

alfalfa leaf protoplast-derived cells.

Cells were cultured at different 2,4-D concentra-

tions as indicated with 10 lM SNP and, as a

control treatment, 10 lM SNP subjected to con-

tinuous illumination for 1 day (iSNP) to ensure

complete decomposition before application to

the protoplasts. Cell division frequency was

counted under a microscope (a). The amount

and the histone H1 phosphorylating activity of

the cognate alfalfa p34cdc2 cyclin-dependent kin-

ase (Medsa;CDKA;1,2) was determined at two

different 2,4-D concentrations (0.22 and 1.0 lM,

respectively) and at two time points by using a

specific antibody (h.i.c. means hours in culture)

(b). The viability of 4-day-old cells (c) was eval-

uated following Evans blue staining and their

elongation is characterized by the ratio of the

largest width and length (d). The average data

and standard deviations derived from three

independent experiments are shown. The char-

acteristic cell morphology of 4-day-old alfalfa

leaf protoplast-derived cells in the presence of

various 2,4-D concentrations (0, 0.22 or 1.0 lM)

and either 10 lM SNP or its light-decomposed,

inactive, form (10 lM iSNP) is also shown (e; bars

represent 3 lm). The relative expression of the

somatic embryogenesis-related MsSERK1 gene

normalized to actin gene expression was meas-

ured in 5-day-old cells by real-time quantitative

PCR (f). The legend to the histograms (a, c, d) is

shown in (g).

NO in cell division 851

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 43, 849–860

leaf protoplasts treated with a relatively high dose (10 lM) of

the synthetic auxin 2,4-D form small, isodiametric, cytoplas-

mically dense and asymmetrically dividing cells expressing

embryogenic competence (Bogre et al., 1990; Dudits et al.,

1991; Pasternak et al., 2002). As can be seen in Figure 2(f),

consistent with the ‘embryogenic morphology’ of the cell

colonies, the relative expression of the MsSERK1 gene,

coding for the somatic embryogenesis receptor kinase

(SERK) protein, a putative marker of the embryogenic

capability of alfalfa cells (Nolan et al., 2003), was consider-

ably higher in the 10 lM 2,4-D-treated or 1 lM 2,4-D plus

10 lM SNP-treated cells in comparison with control cells

(1 lM 2,4-D-grown). To follow the fate of protoplast-derived

cells, they were cultured in the presence of 1 or 10 lM 2,4-D

with or without 10 lM SNP or 1 mM L-NMMA, respectively,

for 5 days and than washed and embedded in an agarose-

solidified ‘low-auxin’ [0.36 lM indoleacetic acid (IAA)] med-

ium. Under these conditions, the cells initially cultured in the

presence of 10 lM 2,4-D or 1 lM 2,4-D plus 10 lM SNP could

develop further into pro-embryogenic cell clusters consist-

ing of small, isodiametric cells with dense cytoplasm (see

Figure S2). In parallel cultures with cells initially grown in

the presence of 1 lM 2,4-D or 1 lM 2,4-D plus 10 lM iSNP,

viability was considerably decreased and mostly clusters of

enlarged, elongated cells developed from the protoplast-

derived cells (see Figure S2). However, a few embryogenic-

type cell clusters could be observed in these types of

cultures as well (data not shown).

L-NMMA inhibits the activation but not the progression of

the cell division cycle and delays formation of embryogenic

cells from leaf protoplast

To further investigate the possible involvement of NO in the

formation and division of protoplast-derived cells, freshly

isolated and cultured leaf protoplasts were treated in the

presence of 1 lM 2,4-D with PTIO, a NO scavenger, or

L-NMMA, a known inhibitor of NO-generating enzymes in

animal and plant cells. Figure 3 shows that the frequency of

cells incorporating BrdU into their nuclei, as well as the

amount and the histone H1 phosphorylating activity of the

cognate alfalfa p34cdc2 kinase Medsa;CDKA;1,2, was signifi-

cantly decreased during the third day of culture in the pres-

ence of either 200 lM PTIO or 1 mM L-NMMA (Figure 3a).

The inhibitory effect of L-NMMA could be reverted by

parallel application of low (1–10 lM) concentrations of the

NO donor SNP, indicating that the L-NMMA-mediated

decrease in S-phase frequency and cyclin-dependent kinase

(CDK) activity were indeed NO dependent (Figure 3a).

Interestingly, the inhibitory effect of L-NMMA on the accu-

mulation and activity of CDK was restricted to the first 3 days

of culture (Figure 3b). These were reduced at 36 and 72 but

not at 96 h of culture. At 96 h, CDK activity was even slightly

increased in comparison with control cells.

To gain a more detailed view of how the effect of L-NMMA

on CDK activity depends on the culture period, 24-h-shifted

48-h-long pulses of the inhibitor were applied to protoplast-

derived cells over a 7-day period (Figure 3c). As a parameter

of cell cycle progression, the frequency of S-phase cells was

determined by incorporation of BrdU into nuclear DNA

during the second half of the L-NMMA pulse. The activity of

the Medsa;CDKA;1,2 kinase was also determined at the end

of the 48-h period. It could be observed that if L-NMMA was

present in the medium before 72 h of culture, there was a

significant inhibition of BrdU incorporation as well as

Medsa;CDK;A1,2 activity. The presence of the inhibitor in

the medium from 72–120 h resulted in increased activity of

the CDK. This might be the consequence of a release of cells

from blocked cell cycle progression which might result in a

synchronized re-entry to the cell cycle. If the inhibitor was

added to the cells 96 h after protoplast isolation, or later, no

effect could be observed.

It could also be observed that application of L-NMMA

decreased the frequency of protoplast-derived cells comple-

ting their first cell division by the fourth day of culture (to

approximately 25–30% as compared with control; data not

shown), but promoted their elongation (Figure 4a). It was

also found that 1 mM of the NOS inhibitor L-NMMA inhibited

the formation of the embryogenic competent cell type in the

presence of 10 lM 2,4-D (Figure 4b). The effect of L-NMMA on

formation of embryogenic cells was transient, as indicated by

the expression of the MsSERK1 gene. The relative MsSERK1

transcript level was lower in cells treated with L-NMMA than

in control cells during the first 2 days of culture but signifi-

cantly increased on the third day and returned to a normal

level by the fifth day (Figure 4c). In agreement with this

expression pattern, L-NMMA-treated cells could indeed

develop into embryogenic cell clusters in the presence of

10 lM 2,4-D (see Figure S2), but the development of pro-

embryogenic cell clusters was slightly delayed (embryogenic

cell clusters were smaller on average, data not shown).

The effect of NO on cyclin gene expression in

protoplast-derived cells

In order to further study the effect of NO on the cell cycle

machinery, the expression of A- and D-type Medicago cyc-

lins, which have proposed roles during the early phases of

cell cycle entry (G0/G1 transition; Dahl et al., 1995; Meskiene

et al., 1995), have been tested in treated protoplasts during

the first 2 days of culture. As shown in Figure 5(a), the rel-

ative expression of the A-type cyclin Medsa;CYCA2;1 is sig-

nificantly increased by 10 lM SNP at 12 and 48 h of culture,

but drops back at 24 h. L-NMMA at 1 mM had no significant

effect on the expression of the same gene. The transcript

level of Medsa,CYCD3;1 was slightly decreased by both

treatments at 12 h and was returned to a normal level at 24 h

followed with a five- to eightfold increase at 48 h. Using a

852 Krisztina Otvos et al.

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 43, 849–860

specific antibody, the protein level of Medsa;CYCD5;1, a

strongly interacting cyclin partner of Medsa;CDKA;1,2 in the

yeast two-hybrid system (Meszaros et al., 2000), was also

checked (Figure 5b). Similarly to the Medsa;CYCD3;1 tran-

scription, the level of this cyclin D protein was also reduced

by both treatments at 12 h, reached the control level at 24 h

and increased by 48 h.

NO fails to interfere with cell cycle progression in

exponentially growing alfalfa cell suspensions

As our observations indicated that protoplast-derived cells

became insensitive to the action of L-NMMA on the

third day of culture when the majority of the cells were

progressing through their first cell division cycle, the

responses of continuously dividing de-differentiated cell

cultures to the same compounds were also investigated.

Cells of a fast growing suspension culture from the same

alfalfa genotype used for leaf protoplast isolation were

studied in these experiments.

The NO donor SNP, at concentrations of 10 or 100 lM, had

no effect on frequency of incorporation of BrdU or on the

mitotic accumulation of the protein Medsa;CDKB;2,1 in

exponentially growing suspension-cultured cells (Fig-

ure 6a). It has been previously demonstrated that the

Medsa;CDKB;2,1 protein accumulates preferentially during

the M-phase of the cell cycle (Magyar et al., 1997; Meszaros

et al., 2000).

(a)

(b)

(c)

Figure 3. The effect of the NOS inhibitor L-

NMMA on cell division of alfalfa leaf protoplast-

derived cells.

(a) Incorporation of BrdU and the amount and

activity of Medsa;CDKA;1,2 in 3-day-old proto-

plast-derived cells treated with the NO scavenger

PTIO (200 lM) or the NOS inhibitor L-NMMA

(1 mM) without or together with the NO donor

SNP (1, 10 or 100 lM) from the beginning of the

protoplast culture.

(b) Changes in level and activity of Medsa;CD-

KA;1,2 protein during the first 4 days of proto-

plast culture in the presence of 1 mM L-NMMA.

(c) The effect of culture period-dependent appli-

cation of L-NMMA (1 mM) on cell cycle (S-phase)

progression and Medsa;CDKA;1,2 activity in

alfalfa leaf-protoplast-derived cells. 24-h-shifted

and 48-h-long pulses of 1 mM L-NMMA were

applied to alfalfa leaf protoplast-derived cells as

indicated in the figure. During the second 24-h

period of L-NMMA treatment, 30 lM BrdU was

incorporated into the medium. At the end of the

L-NMMA pulses, frequency of incorporation of

BrdU and histone H1 phosphorylation activity of

the immunoprecipitated Medsa;CDKA;1,2 kinase

complex were determined.

For BrdU incorporation, the average data and

standard deviations derived from three inde-

pendent experiments are shown. Protoplasts

were grown in the presence of 1 lM 2,4-D during

the presented experiments.

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ª Blackwell Publishing Ltd, The Plant Journal, (2005), 43, 849–860

The NOS inhibitor L-NMMA did not influence the cell

division parameters of the same alfalfa cells if applied

between concentrations of 1 and 10 mM. Figure 6(a) shows

that the presence of 10 mM L-NMMA had no effect either on

S-phase-specific incorporation of BrdU into the nuclear DNA

or on mitosis-specific accumulation of the Medsa;CDKB;2,1

protein kinase. The relative DNA content of the cells was also

unchanged in the presence of 10 mM inhibitor (Figure 6b).

In order to be able to decide whether the absence of the

cell cycle/cell division response is due to a general insensi-

tivity of cultured alfalfa cells for NO or not, the same type of

cell cultures were maintained in the absence of iron for

2–3 weeks in order to allow the use of SNP-mediated

accumulation of the ferritin protein as a marker. Ferritin

gene expression has previously been reported to be induced

by iron as well as by SNP via a NO-dependent mechanism in

iron-deprived Arabidopsis cell cultures (Murgia et al., 2002).

Omitting the iron from the medium did not cause any

difference in the growth rate or cell cycle progression of the

alfalfa cells during the 2–3-week culture period as measured

by packed cell volume and flow cytometric analysis of

cellular DNA content (data not shown). In these cultures,

however, the cellular level of the ferritin protein was reduced

to an undetectable level determined by Western blotting

(a) (c)

(b)

Figure 4. The presence of the NOS inhibitor

L-NMMA prevents embryogenic cell formation

from leaf protoplast-derived cells in response to

10 lM 2,4-D.

(a) Elongation (the ratio of the greatest width and

length) of the cells in control and 1 mM L-NMMA-

treated cells were determined on the fourth day

of culture.

(b) The characteristic cell morphology of 4-day-

old protoplast-derived cells in the presence of 1

or 10 lM 2,4-D without or with 1 mM of L-NMMA

are shown (the indicated scale bar is 6 lm).

(c) The relative expression of the MsSERK1 gene

was determined by real-time QRT-PCR during

the first 5 days of culture. The average data and

standard deviations derived from three inde-

pendent experiments are shown.

(a)

(b)

Figure 5. Effect of NO on cyclin gene expression

in leaf protoplast-derived cells.

(a) Real-time QRT-PCR was used to determine

the relative transcript levels of A-type (Medsa;-

CYCA2;1) and a D-type (Medsa;CYCD3;1) cyclins

in cells grown in the presence of 1 mM L-NMMA

(L) and 10 lM SNP (S), respectively. Transcript

levels were normalized to actin gene expression.

The experiments were carried out in triplicate

and the averages with standard deviations are

shown.

(b) The protein level of the D-type cyclin (Med-

sa;CYCD5;1) was determined by Western blot-

ting by an affinity purified anti-C-terminal

peptide antibody. C, control; L, 1 mM L-NMMA;

S, 10 lM SNP.

854 Krisztina Otvos et al.

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 43, 849–860

using a specific polyclonal antiferritin antibody (Figure 6c,

control).

SNP at 100 lM was capable of inducing accumulation of

ferritin protein in the absence of iron that could be reduced

by the parallel application of the NO scavenger PTIO,

indicating that the cells were responsive to SNP-released

NO (Figure 6d). The specificity of the response was further

supported by the observation that 100 lM iSNP was ineffec-

tive in increasing the level of the ferritin protein (data not

shown).

In addition to cell cycle progression, the growth rate was

also followed in treated and untreated suspension cultures

determining the packed cell volume after 4 days of culture.

No significant differences could be observed (data not

shown). However, if stationary phase cells (11 days old)

were subcultured with a medium supplemented with 10 mM

L-NMMA the growth rate was reduced as measured by the

packed cell volume after 5 days (Figure 7a).

To clarify whether suspension-cultured cells re-entering

the cell cycle can respond to treatment with L-NMMA and

SNP in the same way as leaf protoplast, phosphate-starved

cell cultures were investigated. Phosphate starvation is

widely used to synchronize plant cells in the G0/G1 phase of

the cell cycle (e.g. Dahl et al., 1995; Kapros et al., 1992;

Meskiene et al., 1995), and a synchronous re-entry of the

cells into the cell division cycle can be induced by

re-addition of phosphate. In Figure 7(b) it is shown that

by using a double-phosphate starvation protocol S-phase

frequency could be reduced from 5–6% to approximately

1% in the Medicago cell culture used with a parallel

reduction in activity of Medsa;CDKA;1,2 (and the CDK

protein level as well). Re-addition of phosphate resulted in

a rapid increase in both the S-phase frequency and CDK

activity. If the cells were treated with 10 mM L-NMMA in

parallel with phosphate supplementation, the re-entry of

the cells into division cycle was delayed by approximately

3 h. Similar application of 100 lM SNP had no significant

effect on the cells with regard to the above cell cycle

parameters (data not shown).

Discussion

During the last few years it has become evident that NO

serves as a signaling molecule in plants as well as in animals

(Lamattina et al., 2003). The role of NO in orchestrating plant

defense against biotic and abiotic stresses is probably the

best established (Delledonne et al., 1998; Gould et al., 2003;

Neill et al., 2003). However, there are accumulating pieces of

experimental evidence that NO is also involved in the

regulation of plant growth and development (for reviews see

Lamattina et al., 2003; Neill et al., 2003). Among other

things, NO can stimulate overall plant growth (reviewed by

Neill et al., 2003), root (Gouvea et al., 1997), hypocotyl

(Beligni and Lamattina, 2000) and mesocotyl (Zhang et al.,

(a)

(c)

(d)

(b)

Figure 6. Effect of the NO donor SNP and the NOS-inhibitor L-NMMA on cell

cycle progression of exponentially growing suspension-cultured alfalfa cells.

(a) Cultured M. sativa ssp. varia A2 cells were treated with either SNP (10 or

100 lM) or L-NMMA (10 mM) for 24 h starting on the third day following

subculture. Incorporation of BrdU into nuclear DNA (S-phase) and the

accumulation of the mitosis-specific Medsa;CDKB;2,1 kinase (Magyar et al.,

1997) were determined during and at the end of the 24-h period, respectively.

(b) Flow cytometric analysis of relative DNA content in isolated nuclei of

10 mM L-NMMA-treated cells has also been carried out.

(c, d) In order to test the sensitivity of cultured alfalfa cells towards SNP-

released NO, cells were grown on iron-free medium for 2 weeks before

the experiment, which allowed us to monitor the NO-dependent accumu-

lation of the ferritin protein. It is demonstrated (c) that neither 100 mM

FeSO4, 10 mM L-NMMA nor 100 mM SNP/iSNP influenced cell cycle phase

frequency or level of Medsa;CDKB;2,1 protein in these iron-deprived cells.

However, 100 mM FeSO4 or 100 lM SNP (but not iSNP) resulted in

accumulation of ferritin in the same cells, although this response was not

inhibited by L-NMMA. That the SNP-dependent increase in ferritin level

was indeed mediated by NO was shown by the inhibition of this increase

by 200 lM PTIO (d). Experiments were carried out in triplicate (*indicates

two experiments), and the average values with standard deviations are

shown.

NO in cell division 855

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 43, 849–860

2003) elongation and induce the development of adventi-

tious roots in various plant species (Correa-Aragunde et al.,

2004; Pagnussat et al., 2002, 2003). These observations

indicate that NO may, directly or indirectly, affect cell elon-

gation and division, the two basic processes in plant

morphogenesis. There are no reports, however, on the

involvement of NO in the activation and/or the progression

of the cell division cycle in cultured plant cells.

NO is required for and promotes the activation but not the

progression of the cell division cycle in plants

Individual plant cells without cell walls, i.e. protoplasts,

isolated from leaf tissues and cultured in synthetic liquid

media are probably one of the best in vitro experimental

models for studying the activation of cell division in differ-

entiated plant cells. Although they are isolated using

stressful procedures, experimental evidence demonstrates

that properly handled protoplasts quickly recover in culture

and respond to hormonal as well as stress signals in the

same way as intact plant cells (see, for example, Pasternak

et al., 2002; for reviews Feher et al., 2005; Sheen, 2001).

As the S-phase frequency as well as the division rate of

alfalfa leaf protoplast-derived cells was decreased by the

NOS inhibitor L-NMMA and the NO scavenger PTIO and

increased by the NO donor SNP it is very likely that NO is

involved in the signaling cascade that leads to the de-

differentiation and division of leaf cells. That the observed

effects do indeed depend on NO is strengthened by the

observation that the L-NMMA-mediated inhibition of BrdU

incorporation as well as the activity of Medsa;CDKA;1,2

kinase could be reverted by application of SNP. SNP had a

concentration-dependent effect on the frequency of proto-

plast-derived cells entering the S-phase of the cell cycle. SNP

at concentrations of 1 and 10 lM promoted DNA replication

while SNP at 100 lM inhibited DNA replication. Concentra-

tion-dependent action of NO is well known in both plant and

animal cells. Activation of sea urchin egg cells by NO donors

is only possible in a narrow range of concentrations, and

higher concentrations even inhibit sperm cell-mediated

activation (Kuo et al., 2000). A high level of NO has been

reported to inhibit leaf expansion and plant growth whereas

low levels enhance it (Beligni and Lamattina, 2001; Lamat-

tina et al., 2003; Neill et al., 2003; Seregelyes et al., 2003).

During the process of de-differentiation, the loss of

differentiated functions, recovery from isolation stress, cell

wall synthesis and activation of the cell cycle machinery take

place in a serial, although overlapping, manner in the

protoplast-derived cells (for reviews Feher et al., 2003,

2005). It is not clear at what step NO may accelerate the

process. Shifted pulses of application of L-NMMA and

subsequent measurements of BrdU incorporation and CDK

activity indicate, however, that protoplast-derived cells more

than 3 days old are not responsive to the presence of the

NOS inhibitor. This may indicate that an L-NMMA-sensitive

step operates during the first 3 days of the alfalfa leaf

protoplast culture and this step probably precedes the entry

of the cells into the S-phase of the cell cycle.

In order to address this question, the expression of cyclin

genes previously reported to be activated before the S-

phase entry (Dahl et al., 1995) or even at the G0 to G1

transition (Meskiene et al., 1995), have been tested in the

presence of SNP and L-NMMA. The level of protein of a

further D-type cyclin, the strongest interactor with the

Medsa;CDKA;1,2 kinase in the yeast two-hybrid system

(Meszaros et al., 2000), has also been determined.

Based on the results, specific inhibition of cyclin tran-

scription or accumulation by L-NMMA could not be estab-

lished. The increased level of both cyclins in response to L-

NMMA at 48 h might indicate that the treated cells had

already entered into the division cycle. Interestingly, pro-

toplast-derived cells cultured in the presence of L-NMMA

Pac

ked

cell

volu

me

[ml/l

10m

l cul

ture

]

Con

trol

10 m

M

L-N

MM

A

Med

sa;C

DK

A;1

,2

2.01.81.61.41.21.00.80.6

0.20.0

0.4

201612

8

40

Activity

S phasefrequency

[%]

0

PO4– Control 10 mM

L-NMMA

3h 12h6h 3h 12h6h

Protein

(a) (b)

Figure 7. Effect of L-NMMA on cell division reactivation of suspension-cultured cells.

(a) Eleven-day-old stationary-phase cultures of M. ssp. variaA2 cells were subcultured in the absence (control) and in the presence of 10 mM L-NMMA and the packed

cell volume was measured following 5 days of further culture. The initial packed cell volume is indicated by the dashed line (0.95 ml of cells per 10 ml of culture.

(b) S-phase frequency (determined by flow cytometry) and the amount and histone H1 phosphorylating activity of the Medsa;CDKA;1,2 protein were determined in

suspension-cultured cells following phosphate starvation (see Experimental procedures) and phosphate readdition together with or without 10 mM L-NMMA.

856 Krisztina Otvos et al.

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 43, 849–860

also exhibited increased CDK activity and SERK gene

expression at around the third day of culture. It is

interesting to note that peaks of amount of endogenous

IAA and S-phase frequency could be detected on the

second and third days of culture, respectively, and embry-

ogenic competence is also established during this period

in these protoplast-derived cells (see Pasternak et al., 2000,

2002, for review Feher et al., 2005). It might be speculated

that L-NMMA does not block but slows down cell activation

that results in the synchronous accumulation of the cells in

the G1/S-phases. The synchrony seems to be lost after the

S-phase as the cells at this stage become insensitive to the

presence of L-NMMA. This may result in transiently higher

transcript/protein/activity levels of the investigated genes/

proteins during a certain period. A similar response could

be seen in the same type of culture when cell activation

was slowed down by the buffering of the pH of

the medium (Pasternak et al., 2002; T. P. Pasternak and

A. Feher, unpublished data).

To validate the above hypotheses as well as to determine

the exact timing and nature of the process(es) sensitive to

L-NMMA during the de-differentiation and division of leaf

cells requires further experimentation.

In agreement with the above findings described for leaf

protoplast-derived cells, L-NMMA did not inhibit cell cycle

progression or the growth rate of continuously dividing

suspension-cultured cells, but delayed re-entry of these cells

to the cell division cycle after phosphate starvation or after

the subculture of 11-day-old stationary phase cells. SNP at

concentrations of 10–100 lM had no effect on cell cycle

progression in suspension-cultured alfalfa cells, although

these cells could respond to NO as indicated by 100 lM SNP-

induced and PTIO-sensitive ferritin protein accumulation

(Murgia et al., 2002). This observation indicates that the

absence of the cell cycle response was not due to a general

insensitivity of suspension-cultured cells towards NO.

Although no response to SNP could be observed on

phosphate-starved or stationary phase cells, this may only

be the consequence of the experimental conditions used

(e.g. very rapid re-entry of phosphate starved cells to the cell

cycle) that did not allow a proper resolution of cell cycle

events.

In contrast to the above observations in plant cells, NO is a

well-known cytostatic agent and a regulator of the balance

between cell proliferation and cell differentiation during

animal development (Kuzin et al., 2000; Peunova et al.,

1996). However, the site where the cell cycle is actually

stopped in response to NO varies in different cell types (Guo

et al., 1998; Ishida et al., 1997; Pervin et al., 2001; Peunova

et al., 1996; Takagi et al., 1994). Our experiments revealed

that SNP-generated NO did not block cell cycle progression

in cultured alfalfa cells. It cannot be excluded, however, that

NO is involved in the regulation of cell division and

differentiation in whole plants or organs.

The observations that neither dividing leaf protoplasts nor

dividing suspension-cultured cells are responsive to

L-NMMA support the hypothesis that the activity of

L-NMMA-sensitive plant enzymes is not required to sustain

cell division activity in plant cells.

NO is involved in 2,4-D-induced formation of embryogenic

cells from alfalfa leaf protoplasts

The presented data indicate that although NO is not required

for and does not influence cell cycle progression in expo-

nentially dividing cultured plant cells, it may interact with

auxins linking the regulation of cell division to differen-

tiation. Plants exhibit a remarkable developmental plasticity

compared with animals. Somatic plant cells can regain the

ability to divide during a process termed ‘de-differentiation’,

and ‘de-differentiated’ plant cells can ‘re-differentiate’ into

whole plants under suitable conditions. Our experiments

with embryogenic leaf protoplast-derived cells support the

view that NO in concert with auxin can play an important

role during these transitions.

The altered response of protoplast-derived cells to exo-

genous auxin concentration in the presence of L-NMMA or

SNP, and the observation that in the absence of auxin SNP

could not promote the division of protoplast-derived cells,

may indicate that NO alters the auxin sensitivity of the cells

and/or is involved in mediation of the action of auxin during

these processes. Auxin and NO have also been suggested to

share common steps in signal transduction pathways lead-

ing to root elongation (Gouvea et al., 1997) and formation of

adventitious roots (Pagnussat et al., 2002, 2003, 2004). In

cucumber explants, treatment with IAA induced the level of

endogenous NO in the region where the new root meristems

developed. The effect of NO on IAA-induced root formation

was shown to be dependent on intracellular levels of cGMP,

although cGMP-independent pathways may also exist

(Pagnussat et al., 2003, 2004). The downstream events of

the putative NO-dependent signaling cascade leading to

mitotic activation by auxin are unknown. Whether the same

NO-affected signal transduction pathways operate during

the formation of dividing embryogenic cells from leaf

protoplasts and the formation of adventitious root meristem

remains to be discovered.

In addition to affecting the frequency of dividing cells,

both L-NMMA and SNP affected the pathway of auxin

concentration-dependent development of leaf protoplast-

derived cells. It has been previously shown that these cells

can develop either to vacuolized, elongated cells or to small,

isodiametric cells with dense cytoplasm exhibiting embryo-

genic competence (Bogre et al., 1990; Pasternak et al., 2002,

for reviews Dudits et al., 1991; Feher et al., 2003, 2005).

These developmental pathways are dependent on the con-

centration of exogenous auxin (2,4-D) or oxidative stress-

inducing agents (Pasternak et al., 2002). The application of

NO in cell division 857

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 43, 849–860

the NO donor SNP resulted in the formation of embryogenic-

type cells at a low (1 lM) 2,4-D concentration, although this

type of cell otherwise appears at higher auxin concentra-

tions (5–10 lM 2,4-D). The high-level expression of the

MsSERK1 gene (Nolan et al., 2003) and the further develop-

ment of the cells under culture conditions, allowing somatic

embryo formation, verified that application of SNP could

indeed alter the developmental pathway of the auxin-treated

cells.

The MsSERK1 gene is the alfalfa ortholog of the carrot

(Schmidt et al., 1997) and Arabidopsis (Hecht et al., 2001)

SERK genes implicated both in somatic and zygotic embryo-

genesis. SERK gene expression is frequently used as a

marker of embryogenic competence (for review see Feher

et al., 2003) although its elevated expression was also

associated with auxin-induced root formation (Nolan et al.,

2003) and was suggested to be a morphogenic rather than

only an embryogenic marker.

The application of the NOS inhibitor L-NMMA resulted in

the vacuolization and elongation of the cells at a high (10 lM)

2,4-D level in the medium. Embryogenic cell formation,

however, was not stopped but only delayed in the presence

of L-NMMA, as indicated by SERK gene expression and

further development of the cells. This delay was very similar

to the delay observed in the case of entry of the protoplast-

derived cells into the cell division cycle. It is interesting to

note that delaying cell division activity of the same type of

cells by transiently altering the pH of the medium also

resulted in a parallel inhibition of embryogenic cell forma-

tion (Pasternak et al., 2002).

It is well established that 2,4-D-induced acquisition of

embryogenic competence is associated with endogenous

accumulation of IAA in various types of plant cells including

alfalfa leaf protoplasts (for review Feher et al., 2003). Whe-

ther auxin (in this case 2,4-D) treatment is also associated

with an increase in the level of endogenous NO, in a similar

way to what was observed during root meristem formation

(Pagnussat et al., 2002, 2003), remains an interesting ques-

tion to be answered.

Experimental procedures

Cell cultures and treatments

Protoplasts were isolated from 4–6-week-old alfalfa (Medicagosativa L. ssp. varia; embryogenic genotype A2) plants grown in vitroas described elsewhere (Pasternak et al., 2000, 2002). Purified pro-toplasts were washed twice with W5 solution and resuspended at acell density of 105 protoplasts ml)1 in K75 medium supplementedwith 0.4 M glucose, 0.1 M mannitol, 2.28 lM zeatin and 0.22–10 lM

2,4-dichlorophenoxyacetic acid (2,4-D). The NO donors (�)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide (NOR-3), 3-mor-pholinosydnonimine, HCl (SIN-1), N-acetyl-3-(nitrosothio)-D-valine(SNAP) and 4-phenyl-3-furoxancarbonitrile (furoxan) were pur-chased from Calbiochem (Darmstadt, Germany) as the Nitric OxideDonor Set (catalog no. 482675), and were dissolved as indicated

by the supplier. L-NG-monomethyl-L-arginine (L-NMMA; Sigma,St Louis, MO, USA) was dissolved in water and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO; Sigma) in DMSO.Solutions were sterilized by filtration and added to the medium atthe appropriate final concentrations. The effects of sodium nitro-prusside (SNP; Sigma) were dependent on culturing the protoplastsin diffuse light to ensure prolonged delivery of NO to the cells.Strong light in the presence of SNP caused considerable cell death(data not shown).

The A2 cell suspension culture derived from cells of the samealfalfa genotype used for leaf protoplast isolation were subcul-tured weekly in liquid MS medium supplemented with 0.2 lM 2,4-D and 1 lM kinetin. All treatments were carried out at least intriplicate.

The suspension culture of M. sativa ssp. varia A2 cultured cellswas maintained and synchronized by double phosphate starvationas described by Kapros et al. (1992).

Determination of frequency and viability of cell division

Cell viability was determined with Evans blue, a non-permeatingdye, that leaks through ruptured plasma membranes and stain thecontents of dead cells (Baker and Mock, 1994). Cell elongation wasdetermined under a light microscope by an ocular micrometer at500· magnification and was expressed as the ratio of the meas-ured length and width of the cells. At least 40 randomly chosencells were measured. The frequency of cells that had alreadydivided was visually determined by inspecting more than 500 cellswith a bright field microscope. For the determination of S-phasefrequency, cells were cultivated in the presence of 15–30 lM ofbromodesoxyuridine (BrdU) (Amersham Biosciences, Vienna,Austria) for 12–24 h. Immunological detection of BrdU in isolatednuclei was carried out using standard protocols (Amersham Bio-sciences), as described previously in detail by Pasternak et al.(2000, 2002).

Flow cytometry

Nuclei were released from protoplasts or protoplast-derived cellsinto a suitable buffer by gentle pipetting as described by Galbraithet al. (1993). In the case of cultured cells a 30 min pre-treatment bycell-wall-degrading enzymes (cellulase YC 3%, pectinase 1%, mac-erozyme 1%, in a solution containing 5.4 g mannitol, 5.4 g sorbitoland 50 mg CaCl2 in 100 ml at pH 5.3) was applied. Nuclei werestained with propidium iodide (5 lg ml)1) and (5–10) · 103 nucleiwere used for flow cytometric determination of the relative DNAcontent with a FACSCalibur flow cytometer from Becton Dickinson(Franklin Lakes, NJ, USA). Cell cycle analysis was carried out by theModFit� software.

Protein extraction, kinase activity assay and Western

blotting

For protein isolation, cells were frozen in liquid nitrogen and storedat )70�C until analysis. Histone H1 phosphorylating activity of thecognate alfalfa p34cdc2 kinase (Medsa;CDKA;1,2) was determined byimmunoprecipitation as described by Magyar et al. (1997). Westernblotting was carried out using standard protocols, exactly as des-cribed elsewhere (Pasternak et al., 2000, 2002). The primary anti-bodies were prepared against a C-terminal peptide of the alfalfaMedsa;CDKA;1,2 and Medsa;CDKB;2,1 proteins (Magyar et al.,1997) as well as the Medsa;CYCD5;1 protein (Meszaros et al., 2000)

858 Krisztina Otvos et al.

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 43, 849–860

and against the whole purified alfalfa ferritin protein (Deak et al.,1999) and were affinity purified before use.

Gene expression studies

Messenger RNA populations were isolated from approximately thesame number [(200–250) · 105] of protoplast-derived cells using theDynabeads mRNA Direct Kit (Dynal Biotech, Oslo, Norway). ThemRNAs attached to the beads were treated by RNase-free DNase(Promega, Madison, WI, USA) and were thoroughly washed toprevent contamination by genomic DNA. The purity of the mRNAwas verified by PCR using primers for the actin gene (see below) onsevenfold more mRNA that was present in the cDNA mixture usedfor quantitative RT-PCR. No signals could be obtained indicating theabsence of detectable contamination by genomic DNA. First-strandcDNAs were synthesized by RevertAid M-MuLV reverse transcrip-tase in the presence of 40 U of RiboLock ribonuclease inhibitor(Fermentas, Vilnius, Lithuania). The relative expression of theMsSERK1 gene was measured by real-time PCR on the ABI PRISM7700 sequence detection system (Applied Biosystems, Foster City,CA, USA) using the SYBR-green PCR Master Mix from the samesupplier. The MsSERK1 primer sequences were the same as des-cribed by Nolan et al. (2003). The primers to measure the relativetranscript levels of M. sativa cyclins and the actin B gene (TC86577)as endogenous reference were designed by the Primer Expresssoftware (Applied Biosystems): Medsa;CYCA1;2 forward 5¢-TTT-CTTGCACGATGGACATTAGA-3¢, reverse 5¢-GAGGCATAGTGTTCA-AGAGTTGGA-3¢; Medsa;CycD3;1 forward 5¢-TGTTTAAGAAGACC-AAGAATCAAGGA-3¢, reverse 5¢-TGGCAAGAATTCCGACAATG-3¢;actin B forward 5¢-TCCTAGGGCTGTGTTTCCAAGT-3¢, reverse5¢-CATACCGGTGTCATGGTTGG-3¢.

Primer specificity was verified by cloning and sequencing and byend-point dissociation analysis of PCR products. Real time PCRreactions were carried out in triplicate and the results were analyzedusing the comparative threshold cycle (CT) method and averagedaccording to the advices of the manufacturer (Applied Biosystems).The presented data were derived from three independent treat-ments.

Acknowledgements

The presented experiments were partly supported by theHungarian Scientific Research Fund (grants OTKA T34818; OTKAT37910). AF is grateful for the support of the ‘Janos Bolyai’Research Fellowship.

Supplementary Material

The following supplementary material is available for this articleonline:Figure S1. DAF-2DA (a) and 4-AF (b) fluorescence in leaf protoplast-derived alfalfa cells cultured for 3 days under normal cultureconditions (1 lM 2,4-D as exogenous auxin).Figure S2. Effect of SNP (10 lM) or L-NMMA (1 mM) on theformation of embryogenic cell clusters from alfalfa leaf protoplast-derived cells.

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